Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication.

The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2α (α subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT-/-) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2α phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2α pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT-/- cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2α/SG axis and promotes virus replication through targeting PACT. IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2α phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT-/- cells. SGs induced by PPRV infection through the PKR/eIF2α pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2α/SG axis to favor virus replication.

Peste des petits ruminants virus non-structural V and C proteins interact with the NF-κB p65 subunit and modulate pro-inflammatory cytokine gene induction.

Peste des petits ruminants virus (PPRV) is known to induce transient immunosuppression in infected small ruminants by modulating several cellular pathways involved in the antiviral immune response. Our study shows that the PPRV-coded non-structural proteins C and V can interact with the cellular NF-κB p65 subunit. The PPRV-C protein interacts with the transactivation domain (TAD) while PPRV-V interacts with the Rel homology domain (RHD) of the NF-κB p65 subunit. Both viral proteins can suppress the NF-κB transcriptional activity and NF-κB-mediated transcription of cellular genes. PPRV-V protein expression can significantly inhibit the nuclear translocation of NF-κB p65 upon TNF-α stimulation, whereas PPRV-C does not affect it. The NF-κB-mediated pro-inflammatory cytokine gene expression is significantly downregulated in cells expressing PPRV-C or PPRV-V protein. Our study provides evidence suggesting a role of PPRV non-structural proteins V and C in the modulation of NF-κB signalling through interaction with the NF-κB p65 subunit.

Peste Des Petits Ruminants Virus N Protein Is a Critical Proinflammation Factor That Promotes MyD88 and NLRP3 Complex Assembly.

Inflammatory responses play a central role in host defense against invading pathogens. Peste des petits ruminants virus (PPRV) causes highly contagious acute or subacute disease of small ruminants. However, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. Here, we revealed a novel mechanism by which PPRV induces inflammation. Our study showed that PPRV induced the secretion of interleukin 1ß (IL-1ß) by activating the NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, PPRV replication and protein synthesis were essential for NLRP3 inflammasome activation. Importantly, PPRV N protein promoted NF-κB signaling pathway and NLRP3 inflammasome via direct binding of MyD88 and NLPR3, respectively, and induced caspase-1 cleavage and IL-1ß maturation. Biochemically, N protein interacted with MyD88 to potentiate the assembly of MyD88 complex and interacted with NLPR3 to facilitate NLRP3 inflammasome complex assembly by forming an N-NLRP3-ASC ring-like structure, leading to IL-1ß secretion. These findings demonstrate a new function of PPRV N protein as an important proinflammation factor and identify a novel underlying mechanism modulating inflammasome assembly and function induced by PPRV. IMPORTANCE An important part of the innate immune response is the activation of NF-κB signaling pathway and NLPR3 inflammasome, which is induced upon exposure to pathogens. Peste des petits ruminants virus (PPRV) is a highly contagious virus causing fever, stomatitis, and pneumoenteritis in goats by inducing many proinflammatory cytokines. Although the NF-κB signaling pathway and NLRP3 inflammasome play an important role in regulating host immunity and viral infection, the precise mechanism by which PPRV regulates inflammatory responses remains unknown. This study demonstrates that PPRV induces inflammatory responses. Mechanistically, PPRV N protein facilitates the MyD88 complex assembly by directly binding to MyD88 and promotes the NLRP3 inflammasome complex assembly by directly binding to NLRP3 to form ring-like structures of N-NLRP3-ASC. These findings provide insights into the prevention and treatment of PPRV infection.

Peste des Petits Ruminants Virus Upregulates STING to Activate ATF6-Mediated Autophagy.

Peste des petits ruminants virus (PPRV) infection leads to autophagy, and the molecular mechanisms behind this phenomenon are unclear. Here, we demonstrate that PPRV infection results in morphological changes of the endoplasmic reticulum (ER) and activation of activating transcription factor 6 (ATF6) of the ER stress unfolded protein response (UPR). Knockdown of ATF6 blocked the autophagy process, suggesting ATF6 is necessary for PPRV-mediated autophagy induction. Further study showed that PPRV infection upregulates expression of the ER-anchored adaptor protein stimulator of interferon genes (STING), which is well-known for its pivotal roles in restricting DNA viruses. Knockdown of STING suppressed ATF6 activation and autophagy induction, implying that STING functions upstream of ATF6 to induce autophagy. Moreover, the STING-mediated autophagy response originated from the cellular pattern recognition receptor melanoma differentiation-associated gene 5 (MDA5). The absence of MDA5 abolished the upregulation of STING and the activation of autophagy. The deficiency of autophagy-related genes (ATG) repressed the autophagy process and PPRV replication, while it had no effect on MDA5 or STING expression. Overall, our work revealed that MDA5 works upstream of STING to activate ATF6 to induce autophagy. IMPORTANCEPPRV infection induces cellular autophagy; however, the intracellular responses and signaling mechanisms that occur upon PPRV infection are obscure, and whether innate immune responses are linked with autophagy to regulate viral replication is largely unknown. Here, we uncovered that the innate immune sensor MDA5 initiated the signaling cascade by upregulating STING, which is best known for its role in anti-DNA virus infection by inducing interferon expression. We first provide evidence that STING regulates PPRV replication by activating the ATF6 pathway of unfolded protein responses (UPRs) to induce autophagy. Our results revealed that in addition to mediating responses to foreign DNA, STING can cross talk with MDA5 to regulate the cellular stress response and autophagy induced by RNA viruses; thus, STING works as an adaptor protein for cellular stress responses and innate immune responses. Modulation of STING represents a promising approach to control both DNA and RNA viruses.

A Morbillivirus Infection Shifts DC Maturation Toward a Tolerogenic Phenotype to Suppress T Cell Activation.

Viruses have evolved numerous strategies to impair immunity so that they can replicate more efficiently. Among those, the immunosuppressive effects of morbillivirus infection can be particularly problematic, as they allow secondary infections to take hold in the host, worsening disease prognosis. In the present work, we hypothesized that the highly contagious morbillivirus peste des petits ruminants virus (PPRV) could target monocytes and dendritic cells (DC) to contribute to the immunosuppressive effects produced by the infection. Monocytes isolated from healthy sheep, a natural host of the disease, were able be infected by PPRV and this impaired the differentiation and phagocytic ability of immature monocyte-derived DC (MoDC). We also assessed PPRV capacity to infect differentiated MoDC. Ovine MoDC could be productively infected by PPRV, and this drastically reduced MoDC capacity to activate allogeneic T cell responses. Transcriptomic analysis of infected MoDC indicated that several tolerogenic DC signature genes were upregulated upon PPRV infection. Furthermore, PPRV-infected MoDC could impair the proliferative response of autologous CD4+ and CD8+ T cell to the mitogen concanavalin A (ConA), which indicated that DC targeting by the virus could promote immunosuppression. These results shed new light on the mechanisms employed by morbillivirus to suppress the host immune responses. IMPORTANCE Morbilliviruses pose a threat to global health given their high infectivity. The morbillivirus peste des petits ruminants virus (PPRV) severely affects small-ruminant-productivity and leads to important economic losses in communities that rely on these animals for subsistence. PPRV produces in the infected host a period of severe immunosuppression that opportunistic pathogens exploit, which worsens the course of the infection. The mechanisms of PPRV immunosuppression are not fully understood. In the present work, we demonstrate that PPRV can infect professional antigen-presenting cells called dendritic cells (DC) and disrupt their capacity to elicit an immune response. PPRV infection promoted a DC activation profile that favored the induction of tolerance instead of the activation of an antiviral immune response. These results shed new light on the mechanisms employed by morbilliviruses to suppress the immune responses.

Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction.

BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.

Integrated analysis of long-noncoding RNA and circular RNA expression in Peste-des-Petits-Ruminants Virus (PPRV) infected marmoset B lymphocyte (B95a) cells.

Peste-des-Petits-Ruminants (PPR) or goat plague is an important viral disease of sheep and goats caused by the small ruminant morbilli virus or PPR virus (PPRV). Long non coding RNAs (lncRNA) and circular RNAs (circRNA) play a pivotal role in several biological processes including regulation of virus-host interactions. The present study explored the expression of lncRNA, circRNA and their functions in PPRV infected B-lymphocyte (B95a) cells. The results revealed a total of 4531 lncRNA and 2348 circRNA expression in both mock and PPRV infected samples. Analysis of differentially expressed (DE) RNA identified 123 DE-lncRNA and 39 DE-circRNA as significantly dysregulated. Functional analysis of cis-target genes of DE-lncRNA indicated activation of TCF dependent WNT signaling and PKN1 stimulated transcription process. Interactions (sponging) of microRNA (miRNA) revealed 344 DE-lncRNA-miRNA and 93 DE-circRNA-miRNA pairs. The competing endogenous RNA (ceRNA) network of lncRNA/circRNA-miRNA-mRNA in PPRV infected B95a cells was represented by 69 ceRNA pairs. We validated the DE-circRNA by targeted amplification and sequencing of back spliced junctions (BSJs). The present study revealed a profile of lncRNA, circRNA and their potential ceRNA network in PPRV infection. The results provide insight for better understanding of PPRV-host interactions.

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Long noncoding RNA IRF1-AS is associated with peste des petits ruminants infection.

Peste des petits ruminants (PPR) is an acute and highly contagious disease and has long been a significant threat to small ruminant productivity worldwide. However, the molecular mechanism underlying host-PPRV interactions remains unclear and the long noncoding RNAs (lncRNAs) regulation of PPR virus (PPRV) infection has rarely been reported so far. Here, we first demonstrated that PPRV infection can induce an obvious innate immune response in caprine endometrial epithelial cells (EECs) at 48 h post-infection (hpi) with an MOI of 3. Subsequently, we determined that PPRV infection is associated with 191 significantly differentially expressed (SDE) lncRNAs, namely, 137 upregulated and 54 downregulated lncRNAs, in caprine EECs compared with mock control cells at 48 hpi by using deep sequencing technology. Importantly, bioinformatics preliminarily analyses revealed that these DE lncRNAs were closely related to the immune response. Furthermore, we identified a system of lncRNAs related to the immune response and focused on the role of lncRNA 10636385 (IRF1-AS) in regulating the innate immune response. Interestingly, we found that IRF1-AS was a potent positive regulator of IFN-ß and ISG production, which can significantly inhibit PPRV replication in host cells. In addition, our data revealed that IRF1-AS was positively correlated with its potential target gene, IRF1, which enhanced the activation of IRF3 and the expression of ISGs and interacted with IRF3. This study suggests that IRF1-AS could be a new host factor target for developing antiviral therapies against PPRV infection.

Seroprevalence and associated risk factors of peste des petits ruminants among ovine and caprine in selected districts of Afar region, Ethiopia.

BACKGROUND: A Peste des petits ruminant is an acute, highly contagious and economically important transboundary viral disease of small ruminants. Despite the fact that food and agriculture organization and world organization for animal health plan to eradicate the disease by 2030, some studies indicated an increasing seropositivity of PPR infection in sheep and goats in Ethiopia. A cross-sectional study was employed to estimate the seroprevalence of PPR and to assess risk factors during the study period, February to April, 2020. Following purposive selection of the study districts, simple random sampling technique was employed to select individual animal during sample collection. A total of 384 serum samples were collected from apparently healthy sheep and goats. Competitive Enzyme Linked Immunosorbent Assay was used to detect the presence of antibodies against PPR at national veterinary institute. Descriptive statistics, Pearson's chi-square (X2) and logistic regression analysis were used is this study. RESULTS: The overall animal level seroprevalence of PPR virus was found to be 60.15% (n = 231/384) and species level prevalence rate was found to be 38.18% (n = 42) in sheep and 68.98% (n = 189) in goats in the study areas. Among the associated risk factors considered; species, sex, age and herd sizes were significantly associated (P < 0.05) with the disease occurrence. Among the associated risk factors considered in this study, species, sex, age and herd size were found to be statistically associated with the seropositivity of PPR infection. CONCLUSION: The present study finding revealed that a higher seroprevalence of PPR virus infection and this confirms peste des petits ruminant virus is circulating in Afar region. Further studies should be carried out on the entire region to determine PPR seroprevalence and to develop appropriate control and eradication strategies of PPR disease.

Outbreak of Peste des Petits Ruminants among Critically Endangered Mongolian Saiga and Other Wild Ungulates, Mongolia, 2016-2017.

The 2016-2017 introduction of peste des petits ruminants virus (PPRV) into livestock in Mongolia was followed by mass mortality of the critically endangered Mongolian saiga antelope and other rare wild ungulates. To assess the nature and population effects of this outbreak among wild ungulates, we collected clinical, histopathologic, epidemiologic, and ecological evidence. Molecular characterization confirmed that the causative agent was PPRV lineage IV. The spatiotemporal patterns of cases among wildlife were similar to those among livestock affected by the PPRV outbreak, suggesting spillover of virus from livestock at multiple locations and time points and subsequent spread among wild ungulates. Estimates of saiga abundance suggested a population decline of 80%, raising substantial concerns for the species' survival. Consideration of the entire ungulate community (wild and domestic) is essential for elucidating the epidemiology of PPRV in Mongolia, addressing the threats to wild ungulate conservation, and achieving global PPRV eradication.

Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation.

Peste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-ß) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-ß production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.IMPORTANCE Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

Development of vaccines for prevention of peste-des-petits-ruminants virus infection.

Peste des petits ruminants (PPR) is a highly contagious and fatal disease of small ruminants, particularly sheep and goats. This disease leads to high morbidity and mortality of small ruminants, thus resulting in devastating economic loss to the livestock industry globally. The severe disease impact has prompted the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) to develop a global strategy for the control and eradication of PPR by 2030. Over the past decades, the control of PPR is mainly achieved through vaccinating the animals with live-attenuated vaccines, e.g., rinderpest vaccines. As a closely related disease to PPR of large ruminants, rinderpest was eradicated in 2011 and its vaccines subsequently got banned in order to keep rinderpest-free zones. Consequently, it is desirable to develop homologous PPR vaccines to control the disease. The present review summarizes the objectives of PPR control and eradication by focusing on the homologous PPR vaccines.

Neuropathology mediated through caspase dependent extrinsic pathway in goat kids naturally infected with PPRV.

Peste des petits ruminant (PPR), a highly contagious viral disease of small ruminants, is characterized by erosive stomatitis and pneumo-enteritis. However, its neurovirulence potential as observed with other morbilliviruses has not been fully investigated. The present study describes the neuropathological alterations induced by PPR virus through apoptotic pathway. A total number of 12 carcasses of local breed goat kids of either sex were received for postmortem examination. The clinical history was described as symptoms of mucopurulent nasal discharge, high to low grade fever, erosive stomatitis, dyspnoea and profuse watery diarrhoea followed by mortality of 35 goat kids within a week. The pathoanatomical lesions and immunohistochemical demonstration of PPRV antigen in lungs, intestine, spleen and lymph nodes confirmed PPR disease in goats. Grossly, five brain specimens showed moderate to severe leptomeningeal congestion during necropsy. Microscopically, brain sections showed leptomeningitis and nonsuppurative encephalitis characterized by vascular congestion, haemorrhages in the parenchyma, perivascular cuffing with mild to moderate mononuclear cells (mainly lymphocytes and few macrophages), focal to diffuse microgliosis, neuronal degeneration, satellitosis and neuronophagia. Immunolabelling of viral antigen was observed in the cytoplasm of neurons and glial cells. The RT-PCR amplification of N gene fragment also confirmed the presence of PPRV in the brain. The strong immunoreactivity of Caspase-3, Caspase-8 and comparatively lower expression of caspase-9 along with the absence of any reactivity for Apaf-1 antigen in the brain sections indicated the role of caspase dependent extrinsic pathway in inducing neuropathological changes. The presence of apoptotic neurons in the brain by TUNEL assay further confirmed the apoptosis and strong immunoreactivity of iNOS in neurons which suggested the generation of oxidative stress, that might have induced the apoptosis. The overall findings confirm the neurovirulence potential of PPR virus, via the extrinsic pathway of apoptosis, in natural cases of PPR disease in goat kids.

An investigation on the prevalence of peste des petits ruminants in the camels of Sindh, Pakistan.

The present study investigated the status of peste des petits ruminants (PPR) for the first time in the camels of Pakistan. The samples were collected from the camel residing area of Sindh, Pakistan, and analyzed for breeds (Dhatti and Larri), districts (Tharparkar and Umerkot), age (young, adult, and old), and sexes (male and female). The sera samples (n = 200) were analyzed for the detection of antibodies using a competitive enzyme-linked immunosorbent assay (cELISA). Moreover, the nasal and fecal samples were screened for the PPR virus. Finally, the positive nasal and fecal samples were validated using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocapture enzyme-linked immunosorbent assay (Ic-ELISA). The cELISA results showed an overall prevalence of 8.5% PPR in the study area. The camels of Tharparkar (10.9%; 95% confidence interval (CI) 9.2-12.9) showed higher seroprevalence of PPR antibodies than those of Umerkot (5.5%; 95% CI 4.1-7.2). Moreover, the Larri breed exhibited slightly greater resistance against the disease, because the camels of Dhatti breed (9.0%; 95% CI 7.5-11.0) exhibited a numerically higher (p > 0.05) seroprevalence of PPR in comparison with those of Larri breed (7.9%; 95% CI 6.4-9.9). Furthermore, the young and old camels were more susceptible to the disease attack, because the adults (6.3%; 95% CI 5.0-7.8) exhibited significantly (p < 0.05) lower prevalence rate than the young (9.2%; 95% CI 7.6-11.1) and old (10.3%; 95% CI 8.9-11.9) camels. Finally, the results of the Ic-ELISA and HA test established the 8.3 and 8.2% prevalence of PPR antigen in nasal and fecal material samples, respectively, while the RT-PCR results validated the seropositive animals. These findings confirmed that the prevalence of PPRV infection in the camels of the Sindh province of Pakistan hence urged the need to take effective measures for prevention and control of the disease.

Update on Peste des petits ruminants status in South East Nigeria: serological and farmers' awareness investigation, and potential risk factors.

Peste des petits ruminants (PPR) is a highly contagious, trans-boundary viral disease of sheep and goats that have hindered successful small ruminant farming. Its current status in South East Nigeria with respect to its prevalence and farmers' awareness was studied. Three states, Anambra, Ebonyi, and Enugu, were randomly selected for the study. Sera samples from 113 goats and 172 sheep (collected from December 2017 to June 2018) were randomly collected and analysed for the presence of PPRV antibodies, while structured interview schedules were conducted to elicit information on farmers' awareness of the disease and PPR vaccination and use of veterinary services. An overall seroprevalence of 42.5% (121/285) was recorded. The seroprevalence in decreasing order was 62.2% (Enugu), 34.8% (Anambra) and 20.3% (Ebonyi). There was a significant association (X2 = 36.08, df = 2, p = 0.0001) between seroprevalence and the state sampled. Lack of awareness of PPR vaccination among small ruminant farmers, their limited use of veterinary services (38% consult veterinarians) and non-availability of the vaccine at veterinary establishments in the sampled states are potential risk factors of PPR prevalence in South East Nigeria. Consequently, an effective control measure like mass vaccination is recommended for the study area. Also, there is a need for an extension program for stakeholders and farmers in the study area and country on the grave importance and economic benefits of PPR vaccination and the use of veterinary services.

Structure-Guided Identification of a Nonhuman Morbillivirus with Zoonotic Potential.

Morbilliviruses infect a broad range of mammalian hosts, including ruminants, carnivores, and humans. The recent eradication of rinderpest virus (RPV) and the active campaigns for eradication of the human-specific measles virus (MeV) have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of nonhuman morbilliviruses within human populations, we found that peste des petits ruminants virus (PPRV)-the small-ruminant morbillivirus-is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1-the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterized a single amino acid change, mapping to the receptor-binding domain in the PPRV hemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens.IMPORTANCE A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to provide forewarnings of the emergence of these viruses, it is necessary to develop a better understanding of what determines virus host range, often at the genetic and structural levels. In this study, we demonstrated that the small-ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virus's evolution in the field. Of note, this study was undertaken in vitro, without generation of a fully infectious virus with this phenotype.

The large protein 'L' of Peste-des-petits-ruminants virus exhibits RNA triphosphatase activity, the first enzyme in mRNA capping pathway.

Peste-des-petits-ruminants is a highly contagious and fatal disease of goats and sheep caused by non-segmented, negative strand RNA virus belonging to the Morbillivirus genus-Peste-des-petits-ruminants virus (PPRV) which is evolutionarily closely related to Rinderpest virus (RPV). The large protein 'L' of the members of this genus is a multifunctional catalytic protein, which transcribes and replicates the viral genomic RNA as well as possesses mRNA capping, methylation and polyadenylation activities; however, the detailed mechanism of mRNA capping by PPRV L protein has not been studied. We have found earlier that the L protein of RPV has RNA triphosphatase (RTPase), guanylyltransferase (GTase) and methyltransferase activities, and unlike vesicular stomatitis virus (VSV), follows the conventional pathway of mRNA capping. In the present work, using a 5'-end labelled viral RNA as substrate, we demonstrate that PPRV L protein has RTPase activity when present in the ribonucleoprotein complex of purified virus as well as recombinant L-P complex expressed in insect cells. Further, a minimal domain in the C-terminal region (aa1640-1840) of the L protein has been expressed in E. coli and shown to exhibit RTPase activity. The RTPase activity of PPRV L protein is metal-dependent and functions with a divalent cation, either magnesium or manganese. In addition, RTPase associated nucleotide triphosphatase activity (NTPase) of PPRV L protein is also demonstrated. This work provides the first detailed study of RTPase activity and identifies the RTPase domain of PPRV L protein.

Molecular detection and phylogenetic analysis of Peste des petits ruminants virus circulating in small ruminants in eastern Amhara region, Ethiopia.

BACKGROUND: Peste des Petits Ruminants (PPR) is a severe, highly infectious and fatal viral disease of small ruminants. Four lineages of PPR virus have been identified globally based on sequence analysis of the nucleoprotein (N) and fusion (F) gene. The aim of this study was to isolate and genetically characterize recently circulating PPR virus in small ruminants in the eastern Amhara region in Ethiopia. A total of 28 anti-mortem samples (gum debris, nasal and ocular swab) were collected from clinically suspicious animals and examined for the presence of PPRV by a one-step RT-PCR assay. Samples positive with RT-PCR were subjected to isolation of the virus which were subsequently genetically characterized by sequencing of the nucleoprotein (N) gene and phylogenetic analysis of PPR virus (PPRV) strains. RESULTS: Of the 28 clinical samples examined, 46.4% were positive with RT-PCR for viral nucleic acid. The PPRV was successfully isolated on CHS-20 cell line with the ovine signaling lymphocyte activation molecule (SLAM) receptor expressed on the cell surface and confirmed with RT-PCR and IFAT assay. The nucleotide sequence and phylogenetic analysis indicated that the PPRV obtained were clustered genetically with Lineage IV isolates of the virus. CONCLUSION: The successful isolation of the virus and molecular findings of this study confirmed active lineage IV PPRV infections among populations of sheep and goats in eastern Amhara, suggesting risks for potential spread of the disease to currently free areas. Thus, we recommend systematic vaccination to contain outbreaks in affected districts and geographically linked surrounding districts to which the disease could potentially spread due to different epidemiological linkages.